ABSTRACT

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis–cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

中文翻译：

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狂犬病病毒基质蛋白靶向宿主肌动蛋白细胞骨架：蛋白质-蛋白质相互作用分析

摘要

狂犬病病毒 (RABV) 的多功能基质蛋白 (M) 在狂犬病感染的发病机制中起重要作用。鉴定目标宿主中的 M 蛋白相互作用伙伴有助于阐明参与该病毒发病机制的生物学途径和分子机制。在这项研究中，二维远西方印迹 (2D-Far-WB) 技术被应用于寻找大鼠脑干中可能的基质蛋白伙伴。重组 RABV M 在毕赤酵母中表达并被部分纯化。随后，2D-Far-WB 确定了六种大鼠脑干蛋白与质谱法鉴定的重组 M 蛋白相互作用。通过基因本体分析的功能注释确定这些蛋白质参与突触传递过程、代谢过程和细胞形态发生-细胞骨架组织的调节。通过超分辨率共聚焦显微镜验证了病毒 M 蛋白与感染 RABV 的小鼠 Neuro-2a 细胞中选定宿主蛋白的相互作用。分子对接模拟也证明了 RABV M 复合物的形成。然而，免疫共沉淀的进​​一步确认仅对 M-肌动蛋白细胞质 1 相互作用是成功的。我们的研究揭示了肌动蛋白胞质 1 作为 M 蛋白的结合伙伴，